Since recombinant DNA technology was established in 1980s, eukaryotic simple proteins have been widely produced utilizing E. coli, which allows low-cost production. In living bodies in which they naturally occur, many proteins are first synthesized in the forms of inactive, precursor proteins, and then they undergo cleavage by proteolytic enzymes (e.g., removal of the methionine at their amino-terminus (N-terminus)), or other processing, to form mature proteins. Eukaryotic proteins produced by E. coli, which is a prokaryote, however, often have one or more unnecessary amino acid residues, such, as methionine, which originates from the start codon and is left on their N-terminus. A protein having such one or more unnecessary amino acid residues might provoke an antigen-antibody reaction such as anaphylaxis if it is administered to a human as a medicine. Therefore, such unnecessary amino acid residues should be removed.
Among aminopeptidases, which are enzymes having an activity to cleave amino acid residues one by one starting with the amino-terminus (N-terminus) of a protein, various types are known which differ from one another, e.g., in their specificity. Thus, there have been reports on methods for converting precursor proteins to their corresponding mature proteins utilizing aminopeptidases (e.g., Patent document 1, Patent document 2, and Patent document 3).
For example, human growth hormone, when produced using recombinant E. coli, is obtained in the form of a precursor human growth hormone having a methionine residue which is left on its N-terminus, and thus its amino acid sequence goes, starting from the N-terminus, as “Met-Phe-Pro- . . . ” and so on. Among aminopeptidases, those which have a property that their reaction of cleaving peptide bonds stops one residue before proline in a protein can selectively remove methionine alone at the N-terminus of the precursor human growth hormone. It is well known that human mature growth hormone can be obtained from precursor human growth hormone by using such an aminopeptidase, and pharmaceutical preparations containing human mature growth hormone produced by such a method are currently supplied to the pharmaceutical market.
Various aminopeptidases have been known so far which are suitable for removal of N-terminal methionine like the one that is included in human precursor growth hormone (Patent document 4, Patent document 5). One of such aminopeptidases is an aminopeptidase of Vibrio proteolyticus. This enzyme is produced by translation in the form of a preproprotein consisting of four domains (signal peptide, N-terminal propeptide, mature region, and C-terminal propeptide). The aminopeptidase of V. proteolyticus provides advantages that it is found in the culture medium in which V. proteolyticus has been cultured for a certain length of time, and that it then can be easily isolated from the cells by, e.g., centrifugation. However, it has a great disadvantage that because the amount of aminopeptidase found in such a culture medium is small, a large facility would be required in order to produce aminopeptidase as needed by the industry, and this would prove costly. Thus, studies have been carried out to modify the V. proteolyticus aminopeptidase gene to adapt it for expression in E. coli, by, e.g., replacing its secretion signal with PelB, and thus to let the aminopeptidase be produced and secreted by E. coli. However, the production efficiency of such a method has still been at most about threefold in comparison with the case where V. proteolyticus itself is used.
In general, aminopeptidases (AP), after once produced by translation in an inactive form, are converted to active ones, the mechanism of which has not been fully clarified though. It is known that in some species close to V. proteolyticus, AP is converted to its active form through cleavage by some kind of protease (Non-patent document 1, Non-patent document 2). Again, a neutral protease [vibriolysin: nprV] has been discovered in a culture medium of V. proteolyticus (Non-patent document 3, Non-patent document 4, Non-patent document 5). Vibriolysin is an extracellular zinc metalloprotease, which is produced by translation in the form of a peptide chain composed of a signal peptide consisting of amino acids 1-24, N-terminal peptide consisting of amino acids 25-196, and the remaining amino acids 197-609 forming the mature protein. The role of vibriolysin in connection with the production of the aminopeptidease in V, proteolyticus has not been made clear.    [Patent document 1] Japanese Patent Application Publication No. H62-244381    [Patent document 2] Japanese Patent Application Publication No. H62-500003    [Patent document 3] Japanese Patent Application Publication No. 2004-533263    [Patent document 4] WO 86/01229    [Patent document 5] U.S. Pat. No. 5,569,598    [Non-patent document 1] S. Nirasawa et al. (1999) Biochim Biophys Acta. August 17; 1433(1-2):335-42    [Non-patent document 2] Z Z. Zhang et al. (2000) Biochem J. September 15; 350 Pt 3:671-6    [Non-patent document 3] J. Prescott et al. (1976) Methods Enzymol. 45 404-415    [Non-patent document 4] D. Durham (1990) Appl Environ Microbial. August; 56(8):2277-81    [Non-patent document 5] S. Shinoda et al. (2004) Handbook of Proteolytic Enzymes 2nd ed., 399-40